CN116732013A - Cell wall degrading enzymes with antibacterial activity - Google Patents
Cell wall degrading enzymes with antibacterial activity Download PDFInfo
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- CN116732013A CN116732013A CN202210195657.2A CN202210195657A CN116732013A CN 116732013 A CN116732013 A CN 116732013A CN 202210195657 A CN202210195657 A CN 202210195657A CN 116732013 A CN116732013 A CN 116732013A
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- staphylococcus aureus
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
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- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
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- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Agronomy & Crop Science (AREA)
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- Plant Pathology (AREA)
- Virology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a series of cell wall degrading enzymes with antibacterial activity effective for controlling staphylococcus aureus, wherein the cell wall degrading enzymes with antibacterial activity of the present invention are a series of lyase enzymes with novel amino acid sequences. The cell wall degrading enzyme with antibacterial activity can be used for effectively inhibiting staphylococcus aureus, and can be applied to killing staphylococcus aureus in the environment, treating diseases caused by staphylococcus aureus infection and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a series of cell wall degrading enzymes capable of effectively controlling staphylococcus aureus, application of the cell wall degrading enzymes and a production method of the cell wall degrading enzymes.
Background
Staphylococcus aureus is one of the most important pathogens in humans and can cause a variety of diseases such as: skin, soft tissue, wounds, bone and blood infections, toxic shock syndrome, food poisoning, and the like. [ McCaig LF et al Emerg Infect Dis 2006;12:1715-1723.Laureano AC et al Clin Dermatol 2014;32:711-714. BrU ssow H Environ Microbiol 2016;18:2089-2102 ]. It is one of the most common causes of nosocomial infections, and this bacteria has become a serious threat to hospitals. Furthermore, the emergence and increase of antibiotic-resistant bacteria in a clinical setting is alarming, especially methicillin-resistant bacteria. Recent data from the world health organization indicate that methicillin-resistant staphylococcus aureus (MRSA) strains account for over 20% of all infection cases in the world health organization area, but in some countries this ratio reaches 80% (who 2014. Antimicrobial resistance, global report on surveillance 2014.Who, geneva, switzerland);
in addition, staphylococcus aureus is one of the major pathogens of food-borne diseases, producing enterotoxins in humans (LeLoir Y et al 2003. Genet Mol Res 2:63-76.). In 2014, there was a large outbreak of food infections in the European Union, 7.5% of which were contaminated with staphylococcus aureus (EFSA, ECDC. 2016. The European Union summary report on trends andsources of zoonoses, zoonotic agents and food-borne outbreaks in 2014.EFSA J13:4329.). Also, methicillin-resistant staphylococcus aureus infection is a common problem during animal breeding because livestock can not only infect but also spread bacteria (NormannoG et al 2015.Food Microbiol 51:51-56.). It is well known that increased production of multi-drug resistant bacteria (MDR) by abuse of antibiotics in the fields of food, animal farming, etc. leads to current global health crisis. To address this problem, some countries have restricted the use of antibiotics in animal husbandry (Maron DF, et al 2013.Global Health 9:48.).
With the rapid emergence of resistance to classical antibiotics, alternative treatment regimens are urgently needed. To overcome this problem, phages and cell wall lyase extracted from phages have become an alternative method in recent years.
Phages are viruses that infect only bacteria during their life cycle. In most cases, the bacteriolytic life cycle ends with the death of the bacterial cells, making the phage a natural killer for the bacteria. Cleavage can occur by two mechanisms: firstly, phage with single-chain genome codes a cleavage factor for inhibiting bacterial peptidoglycan biosynthesis; second, in double-stranded DNA (dsDNA) phage, release of phage progeny is mediated by two proteins, holin and endolysin, respectively responsible for cell membrane disruption, and once the virion matures within the bacterial cell, the hollow protein holin forms pores in the intracellular membrane, allowing endolysin to enter the cell wall. Subsequently, endolysin degrades peptidoglycan to allow cell lysis by permeation. In addition, phage infection of gram-negative bacterial hosts requires the provision of additional proteins, known as transmembrane proteins, which help break the outer membrane of the gram-negative host cell. (Catalao MJ et al 2013.FEMS MicrobiolRev 37:554-571)
In addition, virion-associated peptidoglycan hydrolases (VAPGHs) are structural components of virions that allow phage genetic material to enter bacterial cells by slightly degrading peptidoglycans to participate in the initial steps of infection; researchers also refer to the phage virion tail-associated cell wall degrading enzyme TAME (Paul VD, et al 2011; 11:226.) and consider that phage particles of all forms each contain TAME associated with tail structure, causing local degradation of the cell wall facilitating phage DNA injection.
Gram-positive dsDNA phage encoded lytic proteins (endolysin and VAPGHs) exhibit common characteristics, all having a modular structure consisting of different functional domains. On the one hand, this structure confers a remarkable substrate specificity to the cleavage protein; on the other hand, this modular structure facilitates the design of new proteins with enhanced antibacterial activity based on the original ones (Oliveira H et al 2013.J Virol 87:4558-4570.). Most staphylococcal phage endolysins have one or two N-terminal catalytic domains and one C-terminal cell wall binding domain (CBD); similar module structure VAPGHs are also composed of one or two catalytic domains, but lack CBD domains; furthermore, no signal peptide or transmembrane domain was found in lysozymes endolysin in staphylococcal phage. (Rodrii guez-Rubio L et al 2013.Crit Rev Microbiol 39:427-434.).
In order to understand the catalytic mechanism of phage-cleaved proteins, it is important to study the structure of the peptidoglycan of the target bacteria. Vollmer W et al have shown that peptidoglycans consist of short peptide cross-linked linear glycan chains consisting of alternating n-acetylglucosamine (GIcNAc) and n-acetylmuramic acid (murmac) residues, beta-1, 4 glycosidic linkages. In Staphylococcus aureus, the D-lactoyl group of each MurNAc residue was replaced by a peptide stem consisting of L-Ala-Glu-Lys-D-Ala. (Vollmer W et al 2008.FEMS Microbiol Rev 32:259-286.). Researchers divide phage-cleaving proteins into N-acetylmuramidase (N-acetylmuramidase), also known as lysozyme and muramidase (lysozymes or muramidases), according to their catalytic active sites; endo-beta-N-acetylglucosaminidases, also known as glucosaminidases; N-acetylmuramyl-L-alanine amidase (N-acetylmuramyl-L-alanine amidase); transglycosylase (transglycosylase) and endopeptidase (endopeptidase), wherein the endopeptidase is further classified as L-alanyl-D-glutamate endopeptidase, and meta-peptide bridge specific endopeptidase. Glucosaminidases, lysozyme and transglycosidases act on the sugar chain moiety, whereas endopeptidases are responsible for cleaving peptide bridges, amidases degrading the amide bond between sugar and peptide. The lytic enzymes of staphylococcal phages rarely contain transglycosylases, with the catalytic domains being mainly the phage lysozyme domain M23 (LYSO-M23), peptidase domain M23 (PET-M23), amidase 2 domain (AMI-2), amidase 3 domain (AMI-3) and cysteine and histidine dependent amidase/peptidase (CHAP), the most common domains (> 74%) (Oliveira et al 2013.J Virol 87:4558-4570.).
The CBD region of lysozymes in staphylococcus aureus phage typically contains SH 3-related domains, the most common of which are sh3_5 and SH3b (13, 15). SH3b domains have been shown to bind peptidoglycan peptide bridges (GrundlingA, et al 2006.J Bacteriol 188:2463-2472.). However, there are also some staphylococcal phage endolysins in which the CBD has no homology to SH3b (Daniel A, et al 2010.AntimicrobAgents Chemother 54:1603-1612.) such as: endolysins derived from the phages phiNM3, phi13, and MW1, etc. Daniel et al believe that phiNM3 CBD may bind to cell wall related carbohydrates rather than peptide bridges. Recently, a novel CBD was found in the endolysin of phage SA97 (LysSA 97), which has only 19% homology with other staphylococcal endolysins stored in the database (Chang Y et al 2017. ApplMicrobiol Biotechnol 101:147-158.).
Most phage endolysins are highly specific for the genus or species they infect, which has significant advantages over classical broad-spectrum antibiotics; however, the interactions of these proteins with their substrates at the molecular level are not completely understood, it is still not clear which molecular fragment determines the specificity, and thus efforts are made to produce novel chimeric proteins by combining domains of different lyase proteins, and it is interesting that the design of chimeric proteins shows good results in improving the lyase proteins. For example, a chimeric protein PRF-119 designed based on Lysk, obtained by fusing the CHAP domain of Lysk endolysin with the SH3b domain of lysostaphin, PRF-119 shows very good activity against the minimum inhibitory concentration MIC of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus 90 0.391ug/mL; also combining the Ply187 endolysin CHAP domain with the CBD (SH 3 b) domain of Lysk endolysin shows a 10-fold increase in specificity over a single CHAP domain (Mao J, et al 2013.FEMS Microbiol Lett 342:30-36.). Similar chimeric proteins are ClyH (CHAP domain and phiNM3 from plasmin in Ply 187)The CBD of lysozyme), clyF (derived from the CHAP domain of plasmin in Ply187 and CBD of plasmin in Ply 2), and hydh5_sh3b (derived from the CHAP domain of HydH5 and the SH3B domain of lysostaphin) (US 9868943B 2) all exhibited higher bacteriostatic activity against staphylococcus aureus than before the combination. From these data we can conclude that the combination of different domains is a powerful tool that can increase the activity and specificity of phage-cleaving proteins.
In conclusion, two types of lytic proteins in phage, endolysin and VAPGHs, have the potential to degrade peptidoglycan, and can be used as a useful antibacterial agent when exogenously added. The most studied endolysins are currently, and the existing endolysins are improved through the combination of different functional modules; VAPGHs are relatively less studied in functional domains, however the development of novel antibacterial phage lytic proteins requires systematic mining of naturally occurring lytic proteins, and analysis of protein structures mimicking individual functional domains is greatly helpful in directed design of mutants with altered activity or substrate specificity, in addition to designing new proteins by modular combination.
Disclosure of Invention
Based on the background, the invention obtains a series of cell wall lyase with different characteristics by excavating new phage lytic proteins, changing the length or mutating the CHAP functional domain of the VAPGHs tail lyase and combining with CBD regions of different sources, increases the diversity of the existing cell wall lyase, widens the use scene, provides ideas for obtaining more and better-quality lyase, and lays a foundation. It is therefore an object of the present invention to provide a cell wall lyase having a good control effect on staphylococcus aureus as an antibiotic replacement.
The invention firstly provides a cell wall degrading enzyme with antibacterial activity, which is characterized in that the cell wall degrading enzyme is obtained by fusing peptidoglycan lyase or a cell wall domain thereof with a cell wall binding domain, wherein the amino acid sequence of the peptidoglycan lyase is shown as SEQ ID NO.1, and the amino acid sequence of the cell wall domain is shown as SEQ ID NO:5, or the combination mutations A36V, G66S, P88N, Q89A and A129Q on the basis of SEQ ID NO:1, or the combination mutations R69K, P88N, Q89A, Y113W and A129Q on the basis of SEQ ID NO:1, or the combination mutations A36V, R69K, A76S, I79V, P88A and Y113W on the basis of SEQ ID NO:5, or the V36N single point mutation.
Preferably, the C-terminal cell wall binding domain of endo-lysozyme is derived from a staphylococcal phage, more preferably having an amino acid sequence as shown in SEQ ID NO. 8
More preferably, the amino acids of the cell wall degrading enzyme with antibacterial activity are shown in SEQ ID N0.9, SEQ ID N0.10, SEQ ID N0.11, SEQ ID N0.12, SEQ ID NO.13 and SEQ ID N0.14, respectively.
The invention thus provides the coding gene for the cell wall degrading enzyme with antibacterial activity.
Further provides an expression vector and a recombinant cell of the coding gene.
The present invention further provides an antibacterial agent comprising the cell wall degrading enzyme having antibacterial activity as an active ingredient.
Preferably for inhibiting staphylococcus aureus. Or provides the disinfectant with the cell wall degrading enzyme with antibacterial activity as an active ingredient, which is used for killing staphylococcus aureus in the environment.
The invention also provides application of the cell wall degrading enzyme with antibacterial activity in preparing a medicament for preventing or treating diseases caused by staphylococcus aureus infection.
The invention further provides application of the cell wall degrading enzyme with antibacterial activity in preparing a medicine or a skin care product for preventing or treating skin infection.
The invention also provides application of the cell wall degrading enzyme with antibacterial activity in preparing skin care products for preventing or treating skin infection.
Preferably, the prophylactic or therapeutic target is a mammal, more particularly a human, primate, bird, cow, horse, goat, cat, sheep, rodent, dog, pig or poultry animal.
According to the invention, through analyzing the functional domain of the phage genome VAPGHs and related homologous proteins, through structure simulation analysis and prediction, mutation is carried out on the phage genome VAPGHs, and simultaneously the phage genome VAPGHs and CBD from different sources are combined, so that a series of lyase chimeric proteins with antibacterial activity are obtained. The invention provides chimeric proteins which exhibit stronger bacteriostatic activity than the CHAP1 functional domain alone, or the corresponding mutants thereof also exhibit stronger bacteriostatic activity; or exhibit better thermal stability; or exhibit substrate specificity; or the antibacterial agent has the equivalent effect of higher antibacterial rate under different pH conditions, so that the antibacterial agent has stronger practical value.
Drawings
Fig. 1: in example 1, the results of the test of the antibacterial effect of the crude CHAP1 protein on Staphylococcus aureus are shown in the graph, wherein B in the graph represents Buffer control, and 1,2 and 3 represent three parallel experiments of the crude CHAP1 protein.
Fig. 2: SDS-PAGE electrophoresis of the purified chimeric proteins of example 2 shows 1-6 of the proteins SA2, SA2-M1, SA1, SA1-M1, SA1-M2, SA1-M3, respectively.
Fig. 3: the results of the test of the antibacterial effect of the crude enzyme solutions of different chimeric proteins on staphylococcus aureus in example 3 are shown in the graph.
Fig. 4: MIC test results for different chimeric proteins in example 4.
Fig. 5: the results of the test of the antibacterial effect of the different chimeric proteins on staphylococcus aureus and staphylococcus epidermidis in example 5 are shown in the figure, wherein A represents the antibacterial effect on staphylococcus aureus and B represents the antibacterial effect on staphylococcus epidermidis.
Fig. 6: FIG. 6 shows the results of comparison of the enzyme activities of chimeric proteins at different pH conditions.
Detailed Description
The invention will be further illustrated with reference to specific examples. Unless otherwise specified, reagents and equipment used in the following examples are commercially available products.
Example 1 analysis of the encoding Gene of VAPGHs lyase to verify its bacteriostatic Activity
Phage KSAP7 has an inhibitory effect on Staphylococcus aureus, and the phage KSAP7 genome was analyzed to find the presence of endolysin and peptidoglycan lyase VAPGHs in the genome. The VAPGHs also have the potential of degrading peptidoglycan, can be used as a useful antibacterial agent when exogenously added, but the activity of the VAPGHs is not systematically researched by singly using the VAPGHs, the amino acid sequence of the VAPGHs is analyzed, and the amino acid sequence of a CHAP domain is obtained by changing the length of the VAPGHs, and the CHAP domain is named as CHAP1 (the amino acid sequence is shown as SEQ ID NO: 1).
First, the CHAP1 amino acid sequence was optimized according to E.coli codons, and the DNA sequence was integrated between the pET30a expression vectors NdeI and XhoI, and the obtained vector was transformed into BL21 (DE 3) expression strain.
The expression strain is transferred to LB culture medium for activation, cultured at 37 ℃, and cultured overnight at 200 rpm after the strain grows to an OD600 of about 0.8, and IPTG is added to a final concentration of 0.5mM at 16 ℃. The cultured overnight bacterial liquid was centrifuged at 10000 rpm to collect the bacterial cells, and 20 mM Tris-HCl,150mM NaCl,10mM CaCl was used 2 Buffer, pH 7.0, to resuspend cells; the ultrasonic cytoclasis instrument breaks cells, the cytoclasis liquid is centrifuged for more than 30 min at 10000 rpm, and the supernatant is collected as crude protease liquid.
The protein crude enzyme solution is taken as a test object, the antibacterial effect on the staphylococcus aureus GIM1.481 strain is judged, and the test is carried out by referring to a 5.1.1 suspension quantitative antibacterial experiment in WS/T650-2019 standard, and the specific steps are as follows: first with 20 mM Tris-HCl,150mM NaCl,10mM CaCl 2 Staphylococcus aureus cultured overnight at 37℃in LB medium was diluted 1000-fold with Buffer (abbreviated as Buffer) at pH 7.0, added to 50ug/mL of protein solution in an amount of 2% (v/v), incubated at 37℃for 1 hour, and then plated (LB medium) by 10-fold dilution with the above buffers, respectively. The negative control was plated by dilution of staphylococcus aureus broth with buffer to the corresponding gradient. The growth of staphylococcus aureus was observed and compared. The experimental results are shown in Table 1, and the amount of Staphylococcus aureus on the negative control plate is 10 3 About, the colony number of the sample plate treated by the CHAP1 protein crude enzyme solution is not obviously reduced, and the results of three parallel experiments are consistent, which shows that the CHAP1 protein is applied to the golden yellow grape ballsThe bacteria have no remarkable antibacterial activity, and the corresponding graph of the plate antibacterial effect is shown in figure 1.
TABLE 1 number of Staphylococcus aureus colonies in plates
| Sample of | Colony number (number) |
| Negative control | >10 3 |
| CHAP1 parallel experiment 1 | >10 3 |
| CHAP1 parallel experiment 2 | >10 3 |
| CHAP1 parallel experiment 3 | >10 3 |
EXAMPLE 2 analysis of CHAP 1-related homologous proteins, construction of different chimeric proteins with bacteriostatic Activity
As proved by the CHAP1 has no activity, NCBI-Blast sequence alignment is performed on the basis of the CHAP1 amino acid sequence, and the homology of each amino acid sequence is found to be more than 90% compared with other naturally-occurring CHAP domains, the region conservation is stronger, the genetic diversity is low, and most of the functions of the natural CHAP domains are not confirmed. In order to study the CHAP functional domain, a CHAP structural domain (CHAP 2, the amino acid sequence of which is shown as SEQ ID NO: 5) which is derived from Staphylococcus phage vB _Ssmaph-Golestan-105-M genome and has the homology lower than 90% with CHAP1 is selected to perform functional study together, and simultaneously, the amino acid sequence change rule of other CHAP homologous proteins with confirmed functions is combined, and site-directed mutagenesis is performed on the basis of the CHAP1 and the CHAP2 through structural simulation analysis and prediction, so that a novel CHAP amino acid sequence is obtained. The CHAP1 has no effect when used alone, and different combinations of the CHAP catalytic domain and the CBD can improve the specificity and the catalytic activity through the synergistic effect. Wherein the related mutation comprises CHAP1 mutant 1 which is combined mutation A36V, G66S, P88N, Q89A and A129Q (the amino acid sequence is shown as SEQ ID NO: 2) based on CHAP 1; CHAP1 mutant 2 is obtained by carrying out combined mutation on the basis of CHAP1, wherein R69K, P88N, Q89A, Y113W and A129Q (the amino acid sequence is shown as SEQ ID NO: 3); CHAP1 mutant 3 is obtained by carrying out combined mutation on the basis of CHAP1, wherein the amino acid sequences of A36V, R69K, A76S, I79V, P88A and Y113W are shown as SEQ ID NO: 4; CHAP2 mutant 1 was subjected to single point mutation of V36N based on CHAP2 (the amino acid sequence is shown in SEQ ID NO: 6).
Firstly, combining a CHAP1 amino acid sequence with a CBD amino acid sequence (the amino acid sequence is shown as SEQ ID NO: 7) derived from lysP108 endolysin, and combining a CHAP2 amino acid sequence with a CBD amino acid sequence (the amino acid sequence is shown as SEQ ID NO: 8) derived from lysostaphin to perform total gene synthesis, wherein the amino acid sequences are respectively named SA1 and SA2 (the amino acid sequences are respectively shown as SEQ ID N0.9 and SEQ ID NO. 13); the DNA sequence was integrated between the pET30a expression vectors NdeI and XhoI according to E.coli codon optimization. Taking the DNA sequence of SA1 as a template, designing a primer to construct 3 CHAP1 mutants, and connecting and integrating the 3 CHAP1 mutants to a target vector pET30a in a one-step recombination mode, wherein the target vectors are respectively named as SA1_M1, SA1_M2 and SA1_M3; the SA1_M1 amino acid sequence is designed to have 5 site-directed mutations of A36V, G66S, P88N, Q89A and A129Q (the amino acid sequence is shown as SEQ ID NO: 10) based on SA 1; the SA1-M2 mutant designs other 5 mutation site combinations to be R69K, P88N, Q89A, Y113W and A129Q (the amino acid sequence is shown as SEQ ID NO: 11) respectively; the SA 1M 3 was designed to have 6 site-directed mutations of A36V, R69K, A76S, I79V, P88A and Y113W (amino acid sequence shown in SEQ ID NO: 12), respectively. Similarly, the DNA sequence of SA2 is taken as a template, a primer is designed to construct 1 CHAP2 mutant, the mutant is connected and integrated on a target vector pET30a in a one-step recombination mode, the mutant is named as SA2-M1, and a mutation site V36N (the amino acid sequence is shown as SEQ ID NO: 14) exists on the basis of the amino acid sequence of SA 2. Specific information is shown in Table 2, and the vectors are respectively transformed into BL21 (DE 3) expression strains for protein expression.
TABLE 2 chimeric protein Module composition of series
| Encoding | CHAP catalytic domain | CBD | Mutation site | Sequence numbering |
| SA1 | CHAP1 | LysP108 endolysin CBD | Wild type | SEQ ID NO:9 |
| SA1-M1 | CHAP1 mutant 1 | LysP108 endolysin CBD | A36V，G66S，P88N，Q89A，A129Q | SEQ ID NO:10 |
| SA1-M2 | CHAP1 mutant 2 | LysP108 endolysin CBD | R69K，P88N，Q89A，Y113W，A129Q | SEQ ID NO:11 |
| SA1-M3 | CHAP1 mutant 3 | LysP108 endolysin CBD | A36V，R69K，A76S，I79V，P88A，Y113W | SEQ ID NO:12 |
| SA2 | CHAP2 | lysostaphin CBD | Wild type | SEQ ID NO:13 |
| SA2-M1 | CHAP2 mutant 1 | lysostaphin CBD | V36N | SEQ ID NO:14 |
Example 3 preparation of a lyase chimeric protein
The expression strain of example 2 was transferred into LB mediumActivating, culturing at 37deg.C, and growing to OD 600 About 0.8, IPTG was added to a final concentration of 0.5mM, and incubated overnight at 16℃and 200 rpm. The bacterial cells were collected by centrifugation at 10000 rpm from the overnight cultured bacterial liquid, and 20 mM Tris-HCl,150mM NaCl,10mM CaCl was used 2 Buffer, pH 7.0, to resuspend cells; the ultrasonic cytoclasis instrument breaks cells, the cytoclasis liquid is centrifuged for more than 30 min at 10000 rpm, and the supernatant is collected as crude protease liquid. Then obtaining purified protein by His-Tag affinity chromatography, determining protein concentration by photometry, detecting purity and size of the protein by SDS-PAGE (polyacrylamide gel electrophoresis) (figure 2), and evaluating purity of purified chimeric proteins with single band and correct size of about 28kDa>95％。
Example 4 crude enzyme Activity measurement
The protein supernatant prepared in example 3 above was used as a test object to determine the expression level and bacteriostatic activity of different chimeric proteins, excluding inactive mutants. Staphylococcus aureus GIM1.481 is used as a bacteriostasis test strain, and is tested by referring to a WS/T650-2019 5.1.1 suspension quantitative bacteriostasis experiment, specifically as follows: first with 20 mM Tris-HCl,150mM NaCl,10mM CaCl 2 Staphylococcus aureus cultured overnight at 37℃in LB medium was diluted 1000-fold with Buffer (abbreviated as Buffer) at pH 7.0, added to 50ug/mL of protein solution in an amount of 2% (v/v), incubated at 37℃for 1 hour, and then plated (LB medium) by 10-fold dilution with the above buffers, respectively. The negative control was plated by dilution of staphylococcus aureus broth with buffer to the corresponding gradient. The growth of staphylococcus aureus was observed and compared.
The experimental results are shown in Table 3, and the amount of Staphylococcus aureus on the negative control plate is 10 3 About, the number of staphylococcus aureus on a sample plate treated by chimeric protein and corresponding mutant crude enzyme liquid is obviously reduced by about 2-3 orders of magnitude (table 3), which proves that the novel chimeric protein and corresponding mutant obtained by the invention have antibacterial activity on staphylococcus aureus, the crude enzyme liquid antibacterial rate reaches more than 99%, and the corresponding plate antibacterial effect is shown as followsShown in fig. 3.
TABLE 3 number of Staphylococcus aureus colonies in plates
| Sample of | Colony number (number) |
| Negative control | >10 3 |
| SA1 | 0 |
| SA1-M1 | 4 |
| SA1-M2 | 0 |
| SA1-M3 | 0 |
| SA2 | 34 |
| SA2-M1 | 0 |
Example 5 MIC test of chimeric proteins
The minimum inhibitory concentration (minimum inhibitory concentration, MIC) of each protein was determined in 96-well plates using the microdilution method (Wiegand Nature Protocol 2008). Protein was diluted separately with MH (B) medium and incubated with 10 in MH (B) medium 6 cfu/ml staphylococcus aureus culture 1:1 to give a final protein concentration of 128ug/ml to 0.25ug/ml, and a bacterial count of 5X 10 in MH (B) medium as a blank 5 cfu/ml. The optical density was measured at 600nm wavelength after incubation of the well plate at 37℃for 18 hours, and the MIC value of the protein was recorded as the lowest protein concentration at which no bacterial growth occurred.
Results display (fig. 4): SA1 (amino acid sequence shown as SEQ ID NO: 9) protein concentration is more than or equal to 2ug/mL, bacterial cells do not grow, and when the concentration is less than 2ug/mL, bacterial cells grow to different degrees, and 2ug/mL is the minimum bacteriostasis of the protein; the minimum inhibitory concentration of the mutant protein SA1-M2 (amino acid sequence shown as SEQ ID NO: 11) of SA1 is unchanged, while the minimum inhibitory concentration of the mutant SA1-M1 (amino acid sequence shown as SEQ ID NO: 10) and SA1-M3 (amino acid sequence shown as SEQ ID NO: 11) is 0.52ug/mL, which is improved by 4 times, which means that the combined mutations A36V, G66S, P88N, Q89A, A129Q and the combined mutations A36V, R69K, A76S, I79V, P88A, Y113W help to improve the activity of the chimeric protein, reduce the minimum inhibitory concentration, and the combined mutations R69K, P88N, Q89A, Y113W and A129Q do not reduce the minimum inhibitory concentration, but do not reduce the activity of the chimeric protein;
compared with chimeric protein SA1, the minimum inhibitory concentration of wild SA2 (amino acid sequence shown as SEQ ID NO: 13) is higher, and when the concentration is more than or equal to 128ug/mL, the situation that the thalli do not grow occurs, and the minimum inhibitory concentration is 128ug/mL. Compared with the wild SA2, the mutant SA2-M1 (the amino acid sequence is shown as SEQ ID NO: 14) has stronger advantage in the minimum antibacterial concentration, which is improved by 64 times compared with the wild SA2, and the minimum antibacterial concentration is 2ug/mL, which indicates that the mutant site V36N has the capability of improving the activity of the chimeric protein. At present, although the minimum inhibitory concentration of the mutant SA2-M1 is not different from that of the wild SA1, according to the result, the V36N mutation site has the potential of further improving the activities of the SA1 and related mutants, and the minimum inhibitory concentration is reduced.
TABLE 4 minimum inhibitory concentration MIC for each chimeric protein
| Sample of | MIC（ug/mL） |
| SA1 | 2 |
| SA1-M1 | 0.5 |
| SA1-M2 | 2 |
| SA1-M3 | 0.5 |
| SA2 | 128 |
| SA2-M1 | 2 |
Example 5 measurement of chimeric protein thermal stability data
The chimeric proteins were treated at 100℃and 80℃and 55℃for 5min, respectively, to evaluate the thermostability, the initial protein concentration was 50ug/mL, the samples were subjected to high temperature treatment, centrifuged to evaluate the residual protein concentration by photometrically measuring the decrease in optical density, and the proportion of the residual protein was calculated, with the specific results shown in Table 5.
From the results, it can be seen that: the chimeric protein SA2 (the amino acid sequence of which is shown as SEQ ID NO: 13) has better thermal stability, and the residual quantity of the protein is still kept 45% after the chimeric protein SA2 is treated for 5min at 100 ℃; the chimeric protein SA1 (the amino acid sequence of which is shown as SEQ ID NO: 9) has relatively poor heat stability, and has more than 80 percent of protein deactivated aggregation precipitation after being treated for 5 minutes at the temperature of more than 55 ℃; other SA1 mutants have improved heat stability to different degrees compared with the wild SA1, especially the SA1-M2 (amino acid sequence shown as SEQ ID NO: 11) mutants are improved by more than 4 times by different temperature treatments, which proves that different combined mutations are beneficial to the improvement of heat stability. The wild chimeric protein SA2 mutant also keeps higher heat stability, is treated for 5min at 100 ℃, and keeps 67% of protein residue which is 1.5 times of the residue of SA2 under the condition, and the mutation site V36N is beneficial to further improving the heat stability.
TABLE 5 chimeric protein thermal stability data
Example 6 chimeric protein specificity assessment
Antibacterial specificity is an important standard for evaluating the application scene of the lysate, and a reference WS/T650-2019 5.1.1 suspension quantitative antibacterial experimental method is used for testing staphylococcus aureus GIM1.481 and staphylococcus epidermidis ATCC12228 respectively, and the specific method is as follows: first with 20 mM Tris-HCl,150mM NaCl,10mM CaCl 2 Staphylococcus aureus and Staphylococcus epidermidis cultured overnight at 37℃in LB medium were diluted 1000-fold with pH 7.0 buffer, added to 50ug/mL of protein solution in an amount of 2% (v/v), incubated at 37℃for 1 hour, and then plated (LB medium) by 10-fold dilution with the above buffers, respectively. Negative controls were plated with buffer to dilute the staphylococcus aureus and staphylococcus epidermidis cultures to the corresponding gradients. Observation of Staphylococcus aureusThe growth of staphylococcus epidermidis was compared.
Experimental results: as shown in Table 6, the antibacterial rate of the chimeric protein and the corresponding mutant to staphylococcus aureus reaches more than 99%, and only a small amount of colony grows or does not grow on the corresponding flat plate; the chimeric protein wild SA1 (amino acid sequence is shown as SEQ ID NO: 9) and SA2 (amino acid sequence is shown as SEQ ID NO: 13) have certain antibacterial activity on staphylococcus epidermidis, but have relatively poorer activity on staphylococcus aureus, have a certain degree of substrate specificity, reduce the colony quantity by only one order of magnitude compared with a control, and have the antibacterial rate of less than 90 percent; the antibacterial effect of the corresponding mutant on staphylococcus epidermidis is obviously reduced, the number of colonies is not changed by orders of magnitude, and the antibacterial rate is low.
The results show that the combination mutation of different sites or the single-site mutation V36N improves the substrate specificity of the chimeric protein to a certain extent, so that the chimeric protein has antibacterial effect on staphylococcus aureus only in a certain concentration range. The experimental results of the corresponding plate bacteriostasis effect are shown in figure 5.
TABLE 6 number of Staphylococcus aureus and Staphylococcus epidermidis colonies in plates
Example 7 comparison of enzyme Activity of chimeric proteins at different pH conditions
The enzyme activity of chimeric proteins under different pH conditions is also an important criterion for assessing the protein application scenario. Firstly, preparing each chimeric protein into protein solutions with different pH values, wherein the protein concentration is 50ug/mL, and the pH values are respectively: 4.5,5.5,6.5,7.5,8.0,9.0, 10.0, reference WS/T650-2019 5.1.1 suspension quantitative bacteriostasis assay bacteriostasis test on Staphylococcus aureus GIM1.481 with 20 mM Tris-HCl,150mM NaCl,10mM CaCl at different pH 2 Buffer served as blank control. The number of staphylococcus aureus growth (a) on the blank control plate and the number of colony growth (B) on the plate with different pH were counted and compared to calculate the antibacterial rate, i.e. antibacterial rate= (a-B)/a 100%, and the result is shown in fig. 6.
The results show that by comparing the enzyme activity data of chimeric proteins at different pH conditions, it is found that: the chimeric protein SA1 (the amino acid sequence is shown as SEQ ID NO: 9) has higher antibacterial rate reaching more than 95% under different pH conditions; the bacteriostasis rate of the corresponding 3 mutants (amino acid sequences are shown as SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO: 12) is reduced by 70% -80% at the neutral or slightly alkaline pH condition, however, the bacteriostasis activity is still higher under the slightly acidic pH condition, and the bacteriostasis rate is more than 90%; although the SA1 mutant combination mutation has reduced antibacterial activity under neutral or slightly alkaline conditions, the combination mutation still has the potential of digging to adapt to strong acid environment. Compared with wild SA1, chimeric protein SA2 (amino acid sequence shown as SEQ ID NO: 13) has higher antibacterial efficiency under weak acid, weak base or neutral conditions, which reaches more than 98%, but the antibacterial efficiency is greatly reduced to 77% when the pH is adjusted to 10. The mutant SA2-M1 (amino acid sequence is shown as SEQ ID NO: 14) corresponding to SA2 shows stronger antibacterial activity under different pH conditions, the antibacterial rate is more than 98.5%, the antibacterial rate is improved by more than 1.2 times compared with 77% of SA2 under the pH10 condition, and the V36N mutation site is indicated to improve the antibacterial activity of the mutant SA2-M1 under the alkaline condition and is a forward mutation.
<110> Baikurui (Shenzhen) Biotech Co., ltd
<120> cell wall degrading enzyme having antibacterial Activity
<160> 14
<210>1
<211>141
<212>PRT
<213> artificial sequence
<400>1
MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYAYNRRGELGIPVPLWGDAADWIGSAKSAGYGVGRTPK
QGACVIWQRGAPGGSPQYGHVAFVEKVLDGGASIFISEHNYATPNGYGTRTIDMSSAIGKGAQFIYDKG141
<210>2
<211>141
<212>PRT
<213> artificial sequence
<400>2
MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYVYNRRGELGIPVPLWGDAADWIGSAKSAGYSVGRTPK
QGACVIWQRGAPGGSNAYGHVAFVEKVLDGGASIFISEHNYATPNGYGTRTIDMSSQIGKGAQFIYDKG141
<210>3
<211>141
<212>PRT
<213> artificial sequence
<400>3
MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYAYNRRGELGIPVPLWGDAADWIGSAKSAGYGVGKTPK
QGACVIWQRGAPGGSNAYGHVAFVEKVLDGGASIFISEHNWATPNGYGTRTIDMSSQIGKGAQFIYDKG141
<210>4
<211>141
<212>PRT
<213> artificial sequence
<400>4
MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYVYNRRGELGIPVPLWGDAADWIGSAKSAGYGVGKTPK
QGSCVVWQRGAPGGSAQYGHVAFVEKVLDGGASIFISEHNWATPNGYGTRTIDMSSAIGKGAQFIYDKG141
<210>5
<211>141
<212>PRT
<213> artificial sequence
<400>5
MSLDSLKKYNGKLPKHDPSFVQPGNRHYKYQCTWYVYNRRGQLGIPVPLWGDAADWIGGAKGAGYGVGKTPK
QGSCVVWQRGVQGGSAQYGHVAFVEKVLDGGKKIFISEHNWATPNGYGTRTIDMSSAIGKNAQFIYDKK 141
<210>6
<211>141
<212>PRT
<213> artificial sequence
<400>6
MSLDSLKKYNGKLPKHDPSFVQPGNRHYKYQCTWYNYNRRGQLGIPVPLWGDAADWIGGAKGAGYGVGKTPK
QGSCVVWQRGVQGGSAQYGHVAFVEKVLDGGKKIFISEHNWATPNGYGTRTIDMSSAIGKNAQFIYDKK 141
<210>7
<211>111
<212>PRT
<213> artificial sequence
<400>7
KTSSASTPATRPVTGSWKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLPAGATIVYDEVCIQA
GHIWIGYNAYNGNRVYCPVRTCQGVPPNHIPGVAWGVFK111
<210>8
<211>97
<212>PRT
<213> artificial sequence
<400>8
TPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSG
QRIYLPVRTWNKSTNTLGVLWGTIK 97
<210>9
<211>252
<212>PRT
<213> artificial sequence
<400>9
MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYAYNRRGELGIPVPLWGDAADWIGSAKSAGYGVGRTPK
QGACVIWQRGAPGGSPQYGHVAFVEKVLDGGASIFISEHNYATPNGYGTRTIDMSSAIGKGAQFIYDKGKTS
SASTPATRPVTGSWKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLPAGATIVYDEVCIQAGHI
WIGYNAYNGNRVYCPVRTCQGVPPNHIPGVAWGVFK 252
<210>10
<211>252
<212>PRT
<213> artificial sequence
<400>10
MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYVYNRRGELGIPVPLWGDAADWIGSAKSAGYSVGRTPK
QGACVIWQRGAPGGSNAYGHVAFVEKVLDGGASIFISEHNYATPNGYGTRTIDMSSQIGKGAQFIYDKGKTS
SASTPATRPVTGSWKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLPAGATIVYDEVCIQAGHI
WIGYNAYNGNRVYCPVRTCQGVPPNHIPGVAWGVFK252
<210>11
<211>252
<212>PRT
<213> artificial sequence
<400>11
Mtlaslekyngklpkhdpnfvqpgnrhykyqctwyaynrrgelgipvplwgdaadwigsaksagygvgktpk
qgacviwqrgapggsnayghvafvekvldggasifisehnwatpngygtrtidmssqigkgaqfiydkgkts
sastpatrpvtgswkknqygtwykpenatfvngnqpivtrigspflnapvggnlpagativydevciqaghi
wigynayngnrvycpvrtcqgvppnhipgvawgvfk 252
<210>12
<211>252
<212>PRT
<213> artificial sequence
<400>12
MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYVYNRRGELGIPVPLWGDAADWIGSAKSAGYGVGKTPK
QGSCVVWQRGAPGGSAQYGHVAFVEKVLDGGASIFISEHNWATPNGYGTRTIDMSSAIGKGAQFIYDKGKTS
SASTPATRPVTGSWKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLPAGATIVYDEVCIQAGHI
WIGYNAYNGNRVYCPVRTCQGVPPNHIPGVAWGVFK 252
<210>13
<211>240
<212>PRT
<213> artificial sequence
<400>13
MSLDSLKKYNGKLPKHDPSFVQPGNRHYKYQCTWYVYNRRGQLGIPVPLWGDAADWIGGAKGAGYGVGKTPK
QGSCVVWQRGVQGGSAQYGHVAFVEKVLDGGKKIFISEHNWATPNGYGTRTIDMSSAIGKNAQFIYDKKLET
PNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQ
RIYLPVRTWNKSTNTLGVLWGTIK 240
<210>14
<211>240
<212>PRT
<213> artificial sequence
<400>14
MSLDSLKKYNGKLPKHDPSFVQPGNRHYKYQCTWYNYNRRGQLGIPVPLWGDAADWIGGAKGAGYGVGKTPK
QGSCVVWQRGVQGGSAQYGHVAFVEKVLDGGKKIFISEHNWATPNGYGTRTIDMSSAIGKNAQFIYDKKLET
PNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQ
RIYLPVRTWNKSTNTLGVLWGTIK 240
Claims (10)
1. A cell wall degrading enzyme with antibacterial activity is characterized by being obtained by fusing peptidoglycan lyase or a cell wall domain thereof with a cell wall binding domain, wherein the amino acid sequence of the peptidoglycan lyase is shown in SEQ ID NO.1, and the amino acid sequence of the cell wall domain is shown in SEQ ID NO:5, or the combination mutations A36V, G66S, P88N, Q89A and A129Q on the basis of SEQ ID NO:1, or the combination mutations R69K, P88N, Q89A, Y113W and A129Q on the basis of SEQ ID NO:1, or the combination mutations A36V, R69K, A76S, I79V, P88A and Y113W on the basis of SEQ ID NO:5, or the V36N single point mutation.
2. The cell wall degrading enzyme with antibacterial activity according to claim 1, wherein the cell wall binding domain is derived from a C-terminal cell wall binding domain which is an endo-lytic enzyme, more preferably it is derived from a staphylococcal bacteriophage, more preferably its amino acid sequence is shown in SEQ ID No. 7, SEQ ID No. 8.
3. The cell wall degrading enzyme with antibacterial activity according to claim 2, wherein the amino acids are shown in SEQ ID N0.9, SEQ ID N0.10, SEQ ID N0.11, SEQ ID N0.12, SEQ ID NO.13, SEQ ID N0.14, respectively.
4. A gene encoding a cell wall degrading enzyme having antibacterial activity according to any one of claims 1 to 3.
5. An expression vector and a recombinant cell containing the coding gene according to claim 4.
6. An antibacterial agent having the cell wall degrading enzyme having antibacterial activity as claimed in any one of claims 1 to 3 as an active ingredient.
7. An antimicrobial or disinfectant according to claim 6 for inhibiting staphylococcus aureus; preferably for the disinfection of staphylococcus aureus in the environment.
8. Use of a cell wall degrading enzyme having antibacterial activity according to any one of claims 1 to 3 in the manufacture of a medicament for the prophylaxis or treatment of a disease caused by staphylococcus aureus infection.
9. Use of a cell wall degrading enzyme having antibacterial activity according to any one of claims 1 to 3 for the preparation of a medicament or skin care product for the prevention or treatment of skin infections.
10. Use of a cell wall degrading enzyme having antibacterial activity according to any one of claims 1 to 3 for the preparation of a skin care product for the prevention or treatment of skin infections, preferably of a subject such as a mammal, more particularly a human, primate, bird, cow, horse, goat, cat, sheep, rodent, dog, pig or poultry animal.
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